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Specific Wetting Probed With Biomimetic Droplets
Laboratoire Nanobioscience et Microsystèmes group - J. Fattaccioli, J. Baudry, F. Brochard-Wyart, N. Henry and J. Bibette
Soft Matter - 4(12) :2334-40 - DOI:10.1039/B806635C - 2008
We have produced emulsion droplets of controlled size and composition coated by ligands, and studied the adhesion of these drops on a solid substrate coated by receptors and polymers. Using transmission, RICM and fluorescence microscopy we assess the size, contact angle and ligand density for each drop. We first show that non-specific interactions significantly enhance the proteins density within the adhesive patch. Then we show that binding within the patch is partially inhibited in good agreement with the hypothesis of an absence of translational diffusion. We confirm that the density of specific bonds sets the adhesive energy and therefore the final contact angle, and finally show that specific binding in our system is always associated with the existence of a positive line tension, which linearly increases with the density of receptors. These experiments describe a new scenario for specific wetting which raises the importance of the coupling between non-specific interactions and specific binding.
A caged retinoic acid for one- and two-photon excitation in zebrafish embryos
Laboratoire Physique des biomolécules - P. Neveu, I. Aujard, C. Benbrahim, T. Le Saux, J.-F. Allemand, S. Vriz, D. Bensimon and L. Jullien
Angew Chem Int Ed Engl. - 47(20) :3744–46 - DOI:10.1002/anie.200800037 - 2008
In situ quantitative measurement of concentration profiles in a microreactor with submicron resolution using multiplex CARS microscopy
Laboratoire Procédés - Plasmas - Microsystèmes - Dawn Schafer, Jeff A. Squier, Jan van Maarseveen, Daniel Bon, Mischa Bonn, Michiel Mu¨ller
JACS - 130(35) :11592-3 - DOI:10.1021/ja804158n - 2008
In situ quantitative imaging of concentration profiles of reactants and products inside a microfluidic reactor is achieved, with submicron spatial resolution with mM sensitivity and on ms time scales, for a given position. The label-free approach relies on quantitative vibrational spectroscopy, using Coherent Anti-Stokes Raman scattering microscopy in a spectrally resolved fashion, and is demonstrated on an elementary acid-base reaction.
A simple and universal tool to remove on-line impurities in mono- or two-dimensional liquid chromatography–mass spectrometry analysis
Laboratoire Spectrométrie de masse biologique et protéomique - Hesse A-M, Marcelo P, Rossier J and Vinh J.
J. Chrom. A - 1189(1-2) :175-82 - DOI:10.1016/j.chroma.2007.12.060 - 2008
Several recurrent problems have always hindered mono-dimensional liquid chromatography-mass spectrometry proteomic analyses. Polymer contamination is a major problem because polymers could co-elute with compounds of interest (peptides). In this case spectral suppression degrades dynamic range and sensitivity. Polyethylene glycol derivatives count among the major contaminants. They are targeted in this work. They are eluted at 35-40% acetonitrile from C18 phase in every single reversed-phase run. Moreover, they are also observed in two-dimensional liquid chromatography in every salt fraction. A simple and robust method is presented here for rapid and efficient on-line removal of these impurities using self-regenerating purification microdevices.
Determination of nanoparticle diffusion coefficients by Taylor dispersion analysis using a capillary electrophoresis instrument
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - d'Orlye F, Varenne A, Gareil P.
J. Chrom. A - 1204(2) :226-32 - DOI:10.1016/j.chroma.2008.08.008 - 2008
The collective diffusion coefficient D(C) of diluted suspensions of positively charged iron oxide maghemite particles was experimentally investigated using a capillary electrophoresis instrument on the grounds of Taylor dispersion theory. Conditions for this approach to be applicable to nanoparticles of mean solid diameter below 10nm were set in this work, enabling precisions on D(C) determination of less than 2% relative standard deviation (RSD). Significantly different D(C) values were thus measured for particle populations differing in solid number mean diameter by only 2 nm. The obtained values were compared to the z-average diffusion coefficient derived from dynamic light scattering (DLS) experiments and used for the calculation of the Stokes radius. The measured diffusion coefficients appeared to be dependent on particle volume fraction and electrolyte ionic strength. These observations were eventually discussed in terms of particle interactions.
Signal enhancement in electronic detection of DNA hybridization
Laboratoire Nanobiophysiques - C. Gentil, G. Philippin, and U. Bockelmann
Phys. Rev. E - 75(1) :011926 - DOI:10.1103/PhysRevE.75.011926 - 2007
Electronic detection of the specific recognition between complementary DNA sequences is investigated. DNA probes are immobilized at different lateral positions on a Poly(L-lysine)-coated surface of an integrated silicon transistor array. Hybridization and field effect detection are done with the solid surface immersed in electrolyte solutions. Differential measurements are performed, where DNA hybridization leads to surface potential shifts between the transistors of the array. We experimentally show that these differential signals of hybridization can be enhanced significantly by changing the salt concentration between hybridization and detection.
Highly Parallel Mix-and-Match Fabrication of Nanopillar Arrays Integrated in Microfluidic Channels for Long DNA Molecule Separation
Laboratoire Nanobioscience et Microsystèmes group - J. Shi, A. P. Fang, L. Malaquin, A. Pepin, D. Decanini, J. L. Viovy and Y. Chen
Applied Physics Letters - 91(15) :153114 - DOI:153114 10.1063/1.2793616 - 2007
We report on a mix-and-match method based on a combination of soft UV nanoimprint lithography, contact optical lithography, and reactive-ion-etch techniques, which is applicable for high throughput manufacturing of nanostructure integrated microfluidic devices. We demonstrate the integration of high density and high aspect ratio nanopillars into microfluidic channels as electrophoresis sieving matrices. As a result, ? DNA and T4 DNA can be separated within a few minutes. By changing the pattern design, the device could be used for separation of other types of molecules.
Microcontact Printing of Living Bacteria Arrays with Cellular Resolution
Laboratoire Nanobioscience et Microsystèmes group - L. P. Xu, L. Robert, O. Y. Qi, F. Taddei, Y. Chen, A.B. Lindner, D. Baigl
Nano Lett. - 7(7) :2068-72 - DOI:10.1021/nl070983z - 2007
Arrays of living bacteria were printed on agarose substrate with cellular resolution using elastomeric stamps with a high aspect ratio generated by reverse in situ lithography (RISL). The printed bacteria reproduced the original stamp patterns with high fidelity and continued growing as in bulk culture. This methodology provides a simple route to any desired bacterial spatial 2D distribution and may be applied to screening as well as to studies of bacteria phenotypic variability, population dynamics, and ecosystem evolution.
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
Laboratoire Physique des biomolécules - T. Lionnet, M. M. Spiering, S. J. Benkovic, D. Bensimon and V. Croquette
Proc. Nat. Acad. Sci. USA - 104(50) :19790–95 - DOI:10.1073/pnas.0709793104 - 2007
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (approximate to 400 bp/s), its unwinding rate extrapolated to zero force is much slower (approximate to 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
Online preconcentration using monoliths in electrochromatography capillary format and microchips
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - V. Augustin, G. Proczek, J. Dugay, S. Descroix, M.C. Hennion
J. Sep. Sci. - 30(17) :2858-65 - PMID:17973277 - 2007
Online preconcentration and separation of analytes using an in situ photopolymerized hexyl acrylate-based monolith stationary phase was evaluated using electrochromatography in capillary format and microchip. The band broadening occurring during the preconcentration process by frontal electrochromatography and during the desorption process by elution electrochromatography was studied. The hexyl acrylate-based monolith provides high retention for neutral analytes allowing the handling of large sample volumes and its structure allows rapid mass transfer, thus reducing the band broadening. For moderately polar analytes such as mono-chlorophenols that are slightly retained in water, it was shown that enrichment factors up to 3500 can be obtained by a hydrodynamic injection of several bed volumes for 120 min under 0.8 MPa with a decrease in efficiency of 50% and a decrease of 30% for the resolution between 2- and 3-chlorophenol. An 8 min preconcentration time allows enrichment factors above 100 for polyaromatic hydrocarbons. The interest of these monoliths when synthesized in microchip is also demonstrated. A 200-fold enrichment was easily obtained for PAHs with only 1 min as preconcentration time, without decrease in efficiency.
Time-course analysis of mouse serum proteome changes following exposure of the skin to ionizing radiation
Laboratoire Spectrométrie de masse biologique et protéomique - Guipaud O, Holler V, Buard V, Tarlet G, Royer N, Vinh J, Benderitter M.
Proteomics - 7(21) :3992-4002 - PMID:17960731 - 2007
Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.
Polyadenylation of a functional mRNA controls gene expression in Escherichia coli
Laboratoire Spectrométrie de masse biologique et protéomique - Joanny G, Le Derout J, Bréchemier-Baey D, Labas V, Vinh J, Régnier P, Hajnsdorf E.
Nucleic Acids Res. - 35(8) :2494-502 - PMID:17395638 - 2007
Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.
In vivo electrochemical detection of nitric oxide in tumor-bearing mice
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Griveau S., Dumezy C, Seguin J., Chabot GG. Scherman D., Bedioui F.
Anal. Chem. - 79(3) :1030-3 - PMID:17263331 - 2007
Interest in elucidating the mechanisms of action of various classes of anticancer agents and exploring the pathways of the induced-nitric oxide (NO) release provides an impetus to conceive a better designed approach to locally detect NO in tumors, in vivo. We report here on the first use of an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mice. In a first step, we performed the electrochemical characterization of a stable electroactive probe, K4Fe(CN)6, directly injected into the liquid microenvironment especially created around the electrode in the tumor. Second, the ability of the inserted electrode system to detect the presence of NO itself in the tumoral tissue was achieved by using the chemically modified Pt/Ir electrode as NO sensor and two NO donor molecules: diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium 1,2-diolate (DEA-NONOate) and (Z)-1-[N-(2-aminopropyl)-N-(2-ammonio propyl)amino]diazen-1-ium 1,2-diolate (PAPA-NONOate). These two NO donor molecules allowed proving the electrochemical detection of (i) directly injected exogenous NO phosphate buffer solution into the tumor (decomposed DEA-NONOate) and (ii) biomimetically induced endogeneous release of NO in the tumoral tissue, upon injection of PAPA-NONOate into the tumor. This approach could be applied to the in vivo study of candidate anticancer drugs acting on the NO pathways.
Frontal analysis capillary electrophoresis hyphenated to electrospray ionization mass spectrometry for the characterization of the antithrombin/heparin pentasaccharide complex
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Fermas S, Gonnet F, Varenne A, Gareil P, Daniel R.
Anal. Chem. - 79(13) :4987-93 - PMID:17536781 - 2007
The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.
Stable Modification of PDMS surface properties by plasma polymerization: Application to the formation of double emulsions in microfluidic systems
Laboratoire Procédés - Plasmas - Microsystèmes - Vanessa Barbier, Michaël Tatoulian, Hong Li, Farzaneh Arefi-Khonsari ,Armand Ajdari and Patrick Tabeling
Langmuir - 22(12) :5230-2 - DOI:10.1021/la053289c - 2006
We describe a method based on plasma polymerization for the modification and control of the surface properties of poly(dimethylsiloxane) (PDMS) surfaces. By depositing plasma polymerized acrylic acid coatings on PDMS, we succeeded to fabricate stable (several days) hydrophilic and patterned hydrophobic/hydrophilic surfaces. We used this approach to generate direct and (for the first time in this material) double emulsions in PDMS microchannels.
Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremond, Laurent Boitard, Jean Baudry, Jerome Bibette
Biomicrofluidics - 12 44106 - https://doi.org/10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is
becoming a widespread method for performing assays and especially in the cell biol-
ogy field. Making a simple and highly portable system for creating emulsion droplets
would help to continue the popularization of such a technique. Also, the ability to
emulsify all the samples would strengthen this compartimenlization technique to han-
dle samples with limited volume. Here, we propose a strategy of droplet formation
that combines a classical flow-focusing microfluidic chip, which could be commer-
cially available, with a standard laboratory adjustable micropipette. The micropipette
is used as a negative pressure generator for controlling liquid flows. In that way, emul-
sification does neither require any electrical power supply nor a cumbersome device
and functions with small liquid volumes. Droplet formation can be easily and safely
performed in places with limited space, opening a wide range of applications espe-
cially in biological laboratory environments with higher level of safety regulations,
i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid vol-
umes, and thus possible time dependent flow conditions, allows to minimize dead
volume while keeping drops’ size homogeneous. A physical characterization
of droplet production and a model that describes the emulsion features, in terms of
drop size and size distribution, are proposed for rationalizing the performances of
the micropipette-powered emulsification process.
Published by AIP Publishing.
Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremonda), Laurent Boitard, Jean Baudry, and Jérôme Bibette
Biomicrofluidics - 44106 - 10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C, Dagkesamanskaya A, Langer K, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
- 169(6) 335-342. - doi: 10.1016/j.resmic.2018.06.002. -
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Laboratoire Colloïdes et Matériaux Divisés - Dagkesamanskaya A, Langer K, Tauzin AS, Rouzeau C, Lestrade D, Potocki-Veronese G, Boitard L, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
J Microbiol Methods. - 147 59-65 - doi: 10.1016/j.mimet.2018.03.001. -
Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Copyright © 2018 Elsevier B.V. All rights reserved.
Hydrophobization of Silica Nanoparticles in Water: Nanostructure and Response to Drying Stress
Laboratoire Colloïdes et Matériaux Divisés - Solenn Moro, Caroline Parneix, Bernard Cabane, Nicolas Sanson , and Jean-Baptiste d’Espinose de Lacaillerie
Langmuir - 33 (19) 4709–4719 - DOI: 10.1021/acs.langmuir.6b04505 -
We report on the impact of surface hydrophobization on the structure of aqueous silica dispersions and how this structure resists drying stress. Hydrophilic silica particles were hydrophobized directly in water using a range of organosilane precursors, with a precise control of the grafting density. The resulting nanostructure was precisely analyzed by a combination of small-angle X-ray scattering (SAXS) and cryo-microscopy (cryo-TEM). Then, the dispersion was progressively concentrated by drying, and the evolution of the nanostructures as a function of the grafting density was followed by SAXS. At the fundamental level, because the hydrophobic character of the silica surfaces could be varied continuously through a precise control of the grafting density, we were able to observe how the hydrophobic interactions change particles interactions and aggregates structures. Practically, this opened a new route to tailor the final structure, the residual porosity, and the damp-proof properties of the fully dried silica. For example, regardless of the nature of the hydrophobic precursor, a grafting density of 1 grafter per nm2 optimized the interparticle interactions in solution in view to maximize the residual porosity in the dried material (0.9 cm3/g) and reduced the water uptake to less than 4% in weight compared to the typical value of 13% for hydrophilic particles (at T = 25 °C and relative humidity = 80%).

638 publications.